Reconstitution of progesterone receptor with heat shock proteins.
Nonactivated chick progesterone receptor from hypotonic tissue extracts exists in a large complex containing the heat shock proteins hsp90 and hsp70 plus additional smaller proteins; activation of receptor to a DNA-binding form involves the dissociation of proteins from the complex. Whereas numerous attempts to reversibly bind components to the activated receptor have been unsuccessful, we now report conditions that promote the reassociation of hsp90 and hsp70 to progesterone receptor. Cytosolic receptor was dissociated from hsp90 and hsp70 by treatment with 0.5 M KCl and 10 mM ATP in the absence of progesterone. It was then purified by binding to immunoaffinity resins. After wash steps, the receptor-resin complex was incubated in rabbit reticulocyte lysate at 30 C, rewashed, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Saturable binding of rabbit hsp90 and hsp70 to chick receptor was found after incubation with reticulocyte lysate; hsp binding was temperature dependent, but not dependent on exogenous ATP. Incubation of dissolved receptor with oviduct cytosol, from which receptor was obtained, or with purified hsp did not result in hsp binding. Furthermore, mixing oviduct cytosol with lysate inhibited hsp reconstitution, suggesting negative factors for hsp binding in oviduct cytosol. The steroid-binding domain of the receptor was required, since no hsp binding was observed in the reconstitution system using a receptor mutant lacking this domain. When the receptor was isolated in the presence of progesterone, reconstitution with hsp90 and hsp70 did not occur. This is consistent with the in vivo effects of progesterone in promoting hsp dissociation.
Smith
D F
DF
Department of Biochemistry and Molecular Biology, Mayo Medical School, Rochester, Minnesota 55905.
Schowalter
D B
DB
Kost
S L
SL
Toft
D O
DO
D000818:Animals; D002460:Cell Line; D002645:Chickens; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism*; D010057:Oviducts / Q000187:drug effects / Q000378:metabolism*; D011374:Progesterone / Q000494:pharmacology; D011817:Rabbits; D011980:Receptors, Progesterone / Q000187:drug effects / Q000378:metabolism*; D012156:Reticulocytes / Q000187:drug effects / Q000378:metabolism*
D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D011980:Receptors, Progesterone; D011374:Progesterone